The present invention relates to a novel process for the production of transgenic organisms or transgenic cells, to transgenic organisms or transgenic cells obtainable by the process of the present invention, to the use of vectors comprising DNA encoding a recombination promoting enzymes for curing impairments caused by environmental influences in plants or plant cells and for gene therapy in mammals or mammalian cells, and to novel vectors.
The process of homologous recombination requires search for homology, recognition of sequence similarity, and strand exchange between two DNA molecules. In bacteria, these different steps are mediated by a single protein, the RecA protein (for review see:Roca and Cox, 1990), which plays a central role in the recombination pathway of E. coli. However, additional proteins are needed to initiate recombination and to resolve the intermediates created by RecA. Recombination is initiated by the generation of single-stranded DNA (ssDNA) and DNA ends in E. coli and presumably in all organisms. In E. coli, the combined action of the products of the recB, recC, and recD genes initiates a major recombination pathway (for review see: Dunderdale and West, 1994). ssDNA is recognised by RecA protein and double-stranded DNA (dsDNA) is actively searched for. Exchange of complementary strands leads to the formation of recombination intermediates (Holliday structures). The intermediates can be resolved by different pathways; the major one involves the action of the RuvA, RuvB, and RuvC proteins. All of the recombination proteins have to work in concert to complete recombination successfully. Proteins remarkably similar to RecA have been found in a number of eukaryotic cells such as budding yeast, fission yeast, humans, mice, chicken, and plants (Terasawa et al., 1995; for review see: Kowalczykowski and Eggleston, 1994). The best characterised ones are the Dmc1 and Rad51 proteins from Saccharomyces cerevisiae. In both cases the corresponding genes are essential for recombination and the proteins show considerable sequence homology to RecA. A comparison of the primary sequences of Dmc1 and several bacterial RecA proteins suggests that these proteins evolved from a single progenitor before the separation of prokaryotes and eukaryotes. In addition, Rad51 was shown to be structurally very similar to RecA. Rad51 forms DNA/protein filaments, strikingly similar in tertiary structure to those formed with RecA (Ogawa et al., 1993). While previous studies failed to show ATP-dependent homologous pairing and strand-exchange mediated by Rad51 (Shinohara, et al., 1992; Ogawa et al., 1993), more recent experiments have demonstrated these activities (Sung, 1994). Rad51 interacts with other proteins, e.g. Rad52 and Dmc1, so Rad51 may be part of a complex involved in recombination.
However, the complexity of these proteins strongly argues against their being simply a homologue as equivalent to the E.coli RecA protein. Accordingly, different modes of biological activity may be expected.
Various reports have been published focusing on the activity of E. coli RecA protein in animal and in particular in mammalian cells. Thus, Kido et. al, 1992 report on the introduction of functional bacterial RecA protein which was fused to the nuclear location signal of SV40 large T-antigen into mammalian cells. However, no functional studies of the introduced protein were carried out. WO 93/22443 deals with the targeting of exogenous polynucleotide sequences coated on E. coli RecA protein to chromosomal DNA of mammalian cells. This document shows that RecA protein coated oligonucleotides can efficently be targeted to correct chromosomal positions, RecA can stimulate extrachromosomal recombination, and RecA short DNA complexes can be used for gene targeting in mammalian cells. However, the authors failed to show stimulation of homologous recombination in living cells or an entire organism. Spivak et. al (1991) report the increased survival of HeLa cells upon treatment with RecA protein containing liposomes after irradiation. However, RecA stimulated survival was only marginal. Cerruti et al. report on the recombinatorial activity of E.coli RecA protein in plastids which, however, had no effect on DNA repair or cell survival, probably due to the fact that plastids have an own recombination promoting enzyme which is homologous to E.coli RecA. Thus, so far successful experiments with the goal of targeting RecA protein to eukaryotic nuclei which yield a significantly high recombinatorial activity to allow for the industrial applicability of such processes have not been carried out. For example, as regards plant cells, introduction of RecA/DNA complexes, in analogy to WO93/22443, in plant cells by PEG-mediated transformation turned out to be extremely difficult. These complexes exhibit an apparent toxicity and lead to cell death of nearly the total protoplast population. Also, Kido et. al, loc. cit., had reported on the failure to introduce RecA protein into the nuclei of mammalian cells. Therefore, in view of the prior art investigations it was highly questionable as to whether a recombination promoting enzyme such as RecA could be functionally introduced into the nuclei of eukaryotic cells and, furthermore, whether such an introduced RecA protein would indeed be able to enter the cell nucleus and actively promote recombination to an industrially applicable extent.
Thus, the technical problem underlying the present invention was to provide a process for the production of a transgenic organism or a transgenic cell, said process making use of a recombination promoting enzyme. The solution to said technical problem is provided by the embodiments characterised in the claims. Accordingly, the present invention relates to a process for the production of a transgenic organism or a transgenic cell comprising
(a) insertion
(aa) of a DNA into the genome of an organism or a cell, said DNA comprising a DNA which
(aaa) confers to the transgenic organism or the transgenic cell one or more desired characteristics; which
(aab) additionally encodes at least one selection marker expressible in said organism or said cell; and which
(aac) optionally encodes a recombination promoting enzyme or an enzymatically active derivative or part thereof, wherein the recombination promoting enzyme or the enzymatically active part thereof confers the or one of the desired characteristics; or, if (aac) does not apply,
(ab) of a recombination promoting enzyme or an enzymatically active derivative or part thereof in combination with said DNA (aa), into an organism or a cell;
(b) selection of transgenic organisms or cells, which have taken up said DNA or said DNA and said protein according to (a); and
(c) culturing of the desired transgenic organism or the desired transgenic cell in a suitable culture medium.
Thus, it is conceivable in accordance with the present invention that the recombination promoting enzyme confers the desired characteristic or is one of the desired characteristics. In the first instance, the method of the invention may yield, for example, plants with a hyperrecombinant phenotype which might be of use in plant breeding, plants more tolerant to environmental influences, for example, caused by UV or ozone, or plants more tolerant to DNA damage. In the second case, the recombination promoting enzyme may be used to introduce by promoting recombination a DNA sequence of interest into the genome of a cell or an organism. These transgenics are expected to improve the frequency of gene targeting and make this methodology applicable, for example, for plant breeding.
Further, the recombination promoting enzyme may be introduced into the cell or organism as encoded by a corresponding nucleotide sequence which, upon expression, yields said recombination promoting enzyme. Alternatively, the recombination promoting enzyme may be introduced into said cell or organism as such. In this case, the DNA to be inserted encodes a protein with the second or further desired characteristic. The nucleotide sequence encoding the second or further desired characteristics may then be introduced into the genome of said cell or organism by the activity of the recombination promoting enzyme, which, naturally, has to be biologically active in said cell or organism. For example, the second or further characteristic may be an additional protein to be expressed in said cell or organism or, it may be a DNA sequence that, upon recombination into the genome of said organism or said cell, results in the disruption of a naturally occurring or a transgenic gene function. This approach also allows the expression of modified proteins without interference endogenous non-modified copies and allows to circumvent inter-transformant variation. It is also expected that problems arising from instable expression and gene silencing can be circumvented using gene targeting in plants.
For testing the efficacy of the process of the invention, a reproducible and quantitative assay for mitomycin C resistance was developed on the basis of the data and systems published by Lebel et al. (1993). Mitomycin C is known to intercalate in vivo into DNA leading to cross-linking of complementary strands (Borowy-Borowski et al., 1990). Cross-linking leads to inhibition of DNA synthesis in bacteria without concomitant effect on RNA or protein synthesis (Iyer and Szybalski, 1963). In Ustilago maydis and Saccharomyces cerevisiae, mitomycin C was shown to stimulate homologous recombination without being mutagenic (Holliday, 1964). Similar observations were also made in different higher eukaryotic cells (Suzuki, 1965; Shaw and Cohen, 1964; Wang et al., 1988). The data indicate that mitomycin C efficiently blocks DNA replication. The resultant daughter-strand blocks are thought to be repaired in many organisms by homologous recombination (sister-chromatid exchange) and excision repair. Lebel et al. (1993) showed that mitomycin C stimulates intrachromosomal recombination in plant cells, thus pointing to recombinational repair of mitomycin C lesions in plants.
In accordance with the present invention it was found that high mitomycin C concentrations kill untreated plant cells as a representative of eukaryotic cells efficiently, presumably because the capacity of the endogenous repair/recombination system is exhausted. Thus it was further found for the wild-type that protoplast survival under mitomycin C treatment followed a dose-response curve similar to those frequently seen with bacteria and yeast: a shoulder at low doses and a semi-logarithmic decrease at higher doses (FIG. 7). Evaluation of the dose-response curves (FIG. 7) as described by Friedberg (1985) suggests that nt-RecA expression obtained in accordance with the process of the present invention provides the cells with the capacity to repair damage caused by up to 50 xcexcg/ml mitomycin C whereas the endogenous repair mechanism in wild-type tobacco protoplasts can only repair damage caused by up to 15 xcexc/ml of mitomycin C.
Thus, plant cells expressing nt-RecA exhibited a considerably higher resistance to this drug. This suggests that RecA can function in plant cells, interacting with or supplementing the endogenous plant recombination machinery. Furthermore, RecA directly stimulated intrachromosomal recombination in plants. On the basis of this data it may be expected that the process of the present invention yields much higher recombination frequencies than any of the processes described by the prior art.
Although the present invention has been illustrated only with regard to plant cells, the teachings disclosed herein apply as well to other eukaryotic cells such as mammalian cells. It is expected that the present invention allows, for the first time, for a recombination efficiency in the nuclei of eukaryotic cells or organisms that leads to an industrially applicable process for the generation of such transgenic cells or organisms.
In a preferred embodiment of the process of the invention, said transgenic organism or transgenic cell is a plant or a plant cell.
In a most preferred process of the present invention said plant or plant cells is or is derived from Nicotiana tabacum or Arabidopsis thaliana. 
A further preferred embodiment of the invention relates to a method wherein said transgenic organism as transgenic cell is a mammal or a mammalian cell, a fungus, a yeast or a bacterium.
In a further preferred process said desired characteristics are stimulation of homologous recombination, enhancement of gene targeting, stimulation of endogenous mechanisms for repair of DNA damage, thus leading to tolerance to various chemical and physical agents (ozone, UV). Additionally, said further desired characteristic may, for example, be an additional protein expressed, which alters the phenotype of the transgenic organism or cell in a desired way such as the expression of additional surface markers or the expression of different/additional metabolic enzymes. Further, said characteristic may lead to the disruption of a naturally occurring gene function in said organism or said cell.
In another preferred process said selection marker is HygR, KmR, PPTR, MtxR or SulR.
The person skilled in the art is well familiar with these selection markers, where HygR stands for hygromycin resistance, KmR stands for Kanamycin resistance, PPTR stands for phosphonotricin (BASTA) resistance, MtxR stands for methotrexate resistance and SulR stands for sulfonamide resistance. The person skilled in the art is, however, able to replace these preferred selection markers by any other one suitable in the process of the invention.
In an additional preferred embodiment of the process of the invention, said recombination promoting enzyme is the E.coli RecA protein.
In a further preferred process of the present invention said derivative of said recombination promoting enzyme is a fusion protein of the E.coli RecA protein and a nuclear targeting sequence.
The experimental data obtained in accordance with this preferred embodiment showed that nt-RecA was able to increase the UV resistance of recA E. coli. The chimeric protein was capable of binding to ssDNA and to catalyse strand-exchange in vitro about as efficiently as RecA. Interestingly, unlike RecA, nt-RecA showed a high ATPase activity in the absence of ssDNA. This ssDNA-independent activity of nt-RecA was stimulated by addition of ssDNA by the same incremental amount as RecA itself. It is presently not known whether these activities result from two different proteins (nt-RecA and a RecA-like degradation product) in the preparation, or is an intrinsic property of nt-RecA. It can also not be totally excluded that ATPase activity of nt-RecA in the absence of ssDNA is due to trace amounts of contaminating ATPase or DNA, which may have escaped detection by gel electrophoresis and staining. However, it is likely that the nt-RecA fusion protein indeed has different ATPase properties. Since the level of ATPase of nt-RecA in the absence of ssDNA is somewhat higher than that of RecA in the presence of ssDNA one might think of nt-RecA as a modified RecA protein which is constitutively activated in a manner which is reminiscent of the RecA441 and RecA730 proteins (Witkin et al., 1982). ATPase activity of RecA seems to serve two different functions: recycling of the enzyme and overcoming nonhomologous regions of DNA (For review see: Kowalczykowski and Eggelston, 1994; Roca and Cox, 1990).
In a most preferred embodiment said nuclear targeting sequence is the T SV40 nuclear targeting sequence.
The SV40 nuclear targeting sequence is well known in the art and need not be described here any further.
In a still further preferred embodiment of the present invention said insertion is mediated via PEG transformation, Agrobacterium transformation, electroporation, particle bombardment, liposome fusion, in planta transformation, calcium phosphate precipitation, or virus infection.
The optimal process employed depends on the taxonomic origin of the organism or cell to be transfected. The person skilled in the art is well aware of which insertion method is best suited to this purpose.
The invention further relates to a transgenic organism or a transgenic cell obtainable by the process of the invention.
Additionally, the invention relates to a vector comprising a DNA encoding a nuclear targeting sequence operatively linked to a DNA encoding a recombination promoting enzyme or an enzymatically active part thereof, at least one selection marker, and, optionally, at least one further DNA encoding a desired characteristic, wherein the nuclear targeting sequence/recombination promoting enzyme fusion protein encoded by said vector has ATPase activity.
Surprisingly, it was found in accordance with the present invention that the nuclear targeting sequence/recombination promoting enzyme fusion protein as exemplified by the fusion protein wherein the nuclear targeting sequence is derived from SV40 and the recombination promoting enzyme is the E.coli RecA protein confers a high ATPase activity. Said activity appears to serve two different functions: First, it appears to recycle the enzyme and secondly, it appears to overcome non-homologous regions of DNA.
The at least one selection marker comprised in said vector which is capable of expressing the various DNA sequences comprised therein, have been already discussed herein above. The same holds true for the further DNAs encoding a desired characteristic which may also be comprised in the vector of the invention.
In a preferred embodiment of said vector, said nuclear targeting sequence is the T SV40 nuclear targeting sequence. Additionally or alternatively, the recombination promoting enzyme is the E.coli RecA protein.
In a most preferred embodiment said vector of the invention is pS/nt-RecA or pEV/nt-RecA. The construction of said vectors is amply described in Example 1. Further details of said vectors are given in FIG. 1.
Further, the invention relates to the use of a vector of the invention or a vector comprising a DNA which confers to a cell to be transformed or transfected therewith one or more desired characteristics; said DNA additionally encoding at least one selection marker expressible in said cell and further encoding a recombination promoting enzyme or an enzymatically active derivative or part thereof, wherein the recombination promoting enzyme or the enzymatically active part thereof confers the or one of the desired characteristics, for curing impairments caused by environmental influences in plants or plants cells. As regards the selection markers, recombination promoting enzymes and insertion processes, preferred embodiments thereof have been described herein above.
In a preferred embodiment of said use, said impairments are caused by damage to DNA, preferably by UV irradiation, ozone, SO2, methylating agents or mutagenic agents.
In a final preferred embodiment of the invention, the vector described herein above is used for gene therapy in mammals or mammalian cells. Such methods for gene therapy are amply discussed in the art so that the technical details are known to or derivable without further ado by the person skilled in the art.